Bl21 de3 plyss cells

A plasmid (kindly provided by M. Green, U. Mass. Med. School) containing the cDNA sequence encoding the mouse Lcn2 gene in pGEX-2T (GE Healthcare) was expressed in E. coli BL21(DE3)pLysS cells (Invitrogen) [21 (link)]. GST-LCN2 fusion protein was purified by affinity chromatography using Glutathione-Sepharose 4B beads (GE Healthcare). Purified GST-LCN2 was incubated with thrombin-agarose (Sigma-Aldrich) to remove the GST tag. The supernatant containing cleaved GST and LCN2 was incubated with Glutathione-Sepharose to absorb the GST. The supernatant containing only LCN2 was collected for in vitro kinase assays.

ApoA-I WT, Milano (R173C) and A164S proteins were produced and purified as previously described^{30 (link),31 (link)}. In short, a bacterial expression system consisting of pEXP-5 plasmid (Novagen) in Escherichia coli strain BL21(DE3) pLysS cells (Invitrogen) was used to produce the apoA-I proteins. His-tagged ApoA-I proteins were purified from bacterial cell lysate by immobilized metal affinity chromatography (His-Trap-Nickel-chelating columns, GE Healthcare) under denaturing conditions (3 M guanidine in phosphate-buffered saline (PBS), pH 7.4). Following binding, an extensive wash with 40 mM imidazole in PBS was performed and proteins were then eluted with 500 mM imidazole in PBS. Imidazole was removed from protein samples by using desalting columns (GE Healthcare) equilibrated with PBS, pH 7.4, and tobacco etch virus (TEV) protease was employed overnight at 4 °C, in the presence of 1 mM DTT, to remove the His-tag from protein samples. At the end of the incubation, a reverse Ni²⁺-column step was employed in order purify the cleaved ApoA-I from TEV protease and the His-tag. The flow-through containing cleaved ApoA-I protein was desalted into McIlvaine Buffer (165 mM Na₂HPO₄, 17.6 mM citrate, pH 7) and stored at 4 °C prior to use. SDS-PAGE and Blue-native gel (Invitrogen) were used for analyses of lipid-free protein and HDL particles.

Wild-type or mutant VP2 and VP5 proteins were expressed by infecting sf9 cells at an MOI of 5 with recombinant baculovirus for 48 h. Cells were lysed in the lysis buffer (50 mM Tris-HCl [pH 8.0], 200 mM NaCl, 1 mM EDTA, 1% NP40, and protease inhibitor cocktail), and the S-tagged protein was purified using the S-protein agarose (Merck Millipore) and eluted with 3 M MgCl₂. It was then desalted with a PD-10 desalting column (GE Healthcare). VP5 ANC (P348-A526) in the pET28 backbone was expressed in BL21(DE3) pLysS cells (Invitrogen) induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h. Cells were lysed in lysis buffer (50 mM NaH₂PO₄ [pH 8.0], 300 mM NaCl, 5 mM imidazole, 1% NP40, and protease inhibitor cocktail), and the lysate was applied to cobalt resin (Sigma) for His-tagged protein purification.

The wild-type αB-crystallin and αBΔ54–61 mutant were expressed and purified as described previously with some modifications [20 (link)]. In brief, the wild-type protein and deletion mutants were expressed in Escherichia coli BL21(DE3)pLysS cells (Invitrogen Corp., Carlsbad, CA, USA) with IPTG (0.5 mM) induction. The cells (from 1 L culture) were lysed in 15 mL of lysis buffer (50 mM Tris-HCl, 2 mM EDTA, 0.1 M NaCl (pH 7.5)) containing 50 µL of protease inhibitor cocktail III (Calbiochem-EMD Millipore Corporation, Billerica, MA, USA), lysozyme (0.1 mg/mL) (Worthington, Lakewood, NJ, USA) and benzonase nuclease (25 units) (Sigma-Aldrich, St. Louis, MO, USA). The lysate was centrifuged at 18,000× g for one hour. The αB-crystallin or its mutant was precipitated by ammonium sulfate (45%) treatment from the supernatant fraction. The protein precipitate was resolubilized in 3 mL of PBS (phosphate buffer saline) and twice purified by gel filtration chromatography on a Hiload 16/60 Superdex G200 column (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA). The fractions containing the purified crystallins were pooled, concentrated, and stored as 3 mg/mL aliquots at −80 °C for further use. The protein concentration was estimated by Bio-Rad protein assay, and the purity of the samples tested by SDS-PAGE was over 95% (Figure 1B).

Human AP4E1 (residues 881–1135^{18 (link)}) and AP4B1 (residues 612–739^{26 (link)}) appendage domains were expressed as GST-fusion proteins in BL21(DE3)pLysS cells (Invitrogen) for 16–20 h at 22 °C after induction with 0.4 mM IPTG. Proteins were purified in 20 mM HEPES pH 7.5, 200 mM NaCl, and 2 mM 2-Mercaptoethanol. Cells were lysed using a disruptor (Constant Systems Limited) and proteins were affinity purified using glutathione sepharose (GE Healthcare). Fusion proteins were eluted in buffer with 30 mM reduced glutathione and further purified by gel filtration on a Superdex S200 preparative column (GE Healthcare).